RESEARCH ARTICLE

The pharmacological properties of 3-arm or
4-arm DOTA constructs for conjugation to
α-melanocyte-stimulating hormone analogues
for melanoma imaging
Masato Kobayashi ID1,2☯*, Toshitaka Kato2☯, Kohshin Washiyama2,3, Masaaki Ihara2,
Asuka Mizutani2, Kodai Nishi4, Leo G. Flores, 2nd5, Ryuichi Nishii6, Keiichi Kawai2,7a1111111111
a1111111111
a1111111111
a1111111111
a1111111111

1 Wellness Promotion Science Center, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa
University, Kanazawa, Japan, 2 Department of Health Sciences, Institute of Medical, Pharmaceutical and
Health Sciences, Kanazawa University, Kanazawa, Japan, 3 Advanced Clinical Research Center,
Fukushima Global Medical Science Center, Fukushima Medical University, Fukushima, Japan, 4 Departmentof Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan,
5 Department of Pediatrics, MD Anderson Cancer Center, Houston, Texas, United States of America,
6 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 7 Biomedical Imaging
Research Center, University of Fukui, Fukui, Japan☯ These authors contributed equally to this work.
* kobayasi@mhs.mp.kanazawa-u.ac.jp

OPEN ACCESS
Citation: Kobayashi M, Kato T, Washiyama K, Ihara
M, Mizutani A, Nishi K, et al. (2019) The
pharmacological properties of 3-arm or 4-arm
DOTA constructs for conjugation to α-melanocytestimulating hormone analogues for melanoma
imaging. PLoS ONE 14(3): e0213397. https://doi.
org/10.1371/journal.pone.0213397
Editor: Gayle E. Woloschak, Northwestern
University Feinberg School of Medicine, UNITEDSTATES

Abstract
Background
Although a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for
conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to
peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides.

Received: November 4, 2018
Accepted: February 20, 2019

Methods

Published: March 22, 2019

Specific activities, radiolabeling efficiencies, and partition coefficients were evaluated using
111
In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For
assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma
and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-αMSH with and without α-MSH as a substrate. The stability was evaluated using mouse liverhomogenates and plasma. Biological distribution and whole-body single photon emission
computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH wereobtained using B16-F1 melanoma-bearing mice.

Copyright: © 2019 Kobayashi et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper.
Funding: This study was partly funded by Grantsin-Aid for Scientific Research from the Japan
Society for the Promotion of Science (Nos.
15K09949 and 16KK0020 for Masato Kobayashi
and 15K15452 for Keiichi Kawai), the Program of
the Network-type Joint Usage/Research Center for
Radiation Disaster Medical Science of Hiroshima

Results
Specific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM
and 90–95%, respectively. The partition coefficients were −0.28 ± 0.03 for 111In-labeled 3arm DOTA-α-MSH and −0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20191 / 144-arm DOTA construct affects the biological distribution

University, Nagasaki University, and Fukushima
Medical University, and the Nakatani Foundation
for measuring technologies in biomedical
engineering. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declaredthat no competing interests exist.

labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH.
111
In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher
accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Althoughthese stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was
higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution,
111
In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation(1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111Inlabeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH
showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-tomuscle ratios than 111In-labeled 3-arm DOTA-α-MSH.

Conclusions
The 4-arm DOTA construct has better chemical properties for peptide radiotracers than the
3-arm DOTA construct.

Introduction
The incidence rate of malignant melanoma has been steadily increasing over the past 40 years.
The 5-year survival rate with stage IV metastatic melanoma is currently less than 20% because
few effective treatments have been established [1,2]. Because survival is associated with an earlier stage at detection and treatment, specific and highly detectable imaging of melanoma
tumors is strongly desired.
The melanocortin 1 receptor (MC1-R) is one of the most targeted melanoma antigens andbelongs to the melanocortin family of G protein-coupled receptors, which consists of five
receptor subtypes, MC1-R to MC5-R. MC1-R is expressed in nearly all primary and metastaticmelanomas [3] and 95% of uveal melanomas [4]. MC1-R is an attractive receptor for molecular-targeted imaging and radionuclide therapy of melanoma. α-melanocyte-stimulating hormone (α-MSH), a tridecapeptide, is an endogenous ligand for the melanocortin family ofreceptors, with subnanomolar binding affinity to MC1-R [5].
The native α-MSH peptide hormone (Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9Gly10-Lys11-Pro12-Val13-NH2) is proteolytically processed from proopiomelanocortin and isprimarily responsible for regulation of skin pigmentation [6, 7]. Alpha-MSH peptides bind theMC1-R selectively with nanomolar to subnanomolar affinities [8, 9]. Although the native αMSH peptide has been directly radiolabeled with radioiodine etc., it shows low specific activity,
low MC1-R affinity [10], and poor stability [11]. The addition of non-natural amino acids
yielded α-MSH analogues with greater affinity and stability [11].
Radionuclide-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)chelate has been conjugated to many peptides and provides better specific activity, MC1-R
affinity, and stability for melanoma imaging of MC1-R [12]. Although the 3-arm DOTA construct, which has three carboxylic acids, has been applied for conjugation to many peptides,
the 4-arm DOTA construct, which has four carboxylic acids, improves radiochemical yield,
specific activity, and stability of therapeutically active conjugates to antibodies for radioimmunotherapy of cancer [13]. In this study, we investigated if the 4-arm DOTA construct conjugated to peptides improves the chemical properties for melanoma imaging of MC1-R
compared to 3-arm DOTA-conjugated peptides. Numerous α-MSH conjugated peptide

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20192 / 144-arm DOTA construct affects the biological distribution

Fig 1. Structures of the 3-arm DOTA construct (a) and 4-arm DOTA construct (b). https://doi.org/10.6084/m9.
figshare.7697861.
https://doi.org/10.1371/journal.pone.0213397.g001

analogues have been developed with high affinities and specificities for α-MSH receptors [14].
Native α-MSH, as an example of a peptide, was selected to evaluate improvement of chemical
properties using the 4-arm DOTA construct because 3-arm DOTA-conjugated native α-MSH(3-arm DOTA-α-MSH, Fig 1A) yielded relatively low MC1-R affinity and stability comparedto other 3-arm DOTA-conjugated peptides [14].

Materials and methods
Reagents and radionuclides
α-MSH free acid was purchased from Abcam (Cambridge, UK). The chelating agent DOTA
and N-hydroxysuccinimidyl (NHS)-ester and S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) were obtained from Macrocyclics, Inc.
(Plano, TX, USA). 111InCl3 was purchased from FUJIFILM RI Pharma Co., Ltd. (Chiba,
Japan).

Synthesis and purification of 3-arm and 4-arm DOTA constructs
The conjugation procedure was performed using the methods of Maguire and Greg et al., withsome modifications [13,15]. Briefly, to form the 3-arm DOTA-α-MSH, α-MSH free acid (1 eq)
was dissolved in purified water (10 mg/mL solution) and conjugated to DOTA-NHS-ester (40eq), which was dissolved in N,N-dimethylformamide (40 mg/mL solution), using 0.1 M
NaHCO3 buffer (pH 7–8) for 6 hours at room temperature.
For 4-arm DOTA-conjugated native α-MSH (4-arm DOTA-α-MSH, Fig 1B), p-SCN-BnDOTA (10 eq) was dissolved in N,N-dimethylformamide (40 mg/mL solution). α-MSH free

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20193 / 144-arm DOTA construct affects the biological distribution

acid (1 eq, 10 μg/mL purified water solution) was conjugated to p-SCN-Bn-DOTA using 0.1
M NaHCO3 buffer (pH 7–8) for 24 hours at room temperature.
These resulting products were purified using high performance liquid chromatography(HPLC, Hitachi, Ibaraki, Japan) using a 5C18-AR-II column (Cosmosil, Nakalai Tesque,
Kyoto, Japan), a combination gamma counter, and 210-nm ultraviolet light. For the 3-arm
DOTA construct, the initial solvent was 0.05% trifluoroacetic acid in water (pH 2.2): acetonitrile at 78:22 after injection, and was gradually changed to 74:26 after 20 min as the eluent at aflow rate of 1.0 mL/min. For the 4-arm DOTA construct, the initial solvent was 0.05% trifluoroacetic acid in water (pH 2.2): acetonitrile at 78:22 after injection, and was gradually changed
to 75:25 after 40 min as the eluent at a flow rate of 1.0 mL/min.

Radiolabeling, purification, and partition coefficients
The 3-arm and 4-arm DOTA-α-MSH were labeled by the addition of 111InCl3 in 1 M
CH3COONH4 buffer (pH 5.5, 100 mL) at 70˚C for 5 min [16,17]. 111In-labeled 3-arm and4-arm DOTA-α-MSH were purified by reversed phase-HPLC. For 3-arm DOTA-α-MSH, theinitial solvent was 0.01% trifluoroacetic acid in water (pH 2.5): acetonitrile at 76:24, and was
gradually changed to 75:25 after 20 min as the eluent at a flow rate of 1.0 mL/min and UV
detector (895 V and 210 nm). For 4-arm DOTA-α-MSH, the solvent was 0.01% trifluoroacetic
acid in water (pH 2.0): acetonitrile at 73:27.
The partition coefficients of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were measured
using 2.0 mL n-octanol as the organic phase and 2.0 mL 0.l M phosphate buffer as the aqueous
phase (pH 7.4 for plasma) [18]. N-octanol and the buffer were pre-mixed twice using amechanical mixer for 1 min at room temperature. Then, 20 υL radioactive sample were added
and mixed twice using a mechanical mixer for 1 min at room temperature. The radioactivityof 200 uL of each phase was measured after centrifugation. Calculation of log (n-octanol/0.l M
phosphate buffer) was performed.

Tumor cells
Mouse skin melanoma B16-F1 cells with high MC1-R expression and mouse breast cancer
4T1 cells with low MC1-R expression were obtained from American Type Culture Collection.
B16-F1 cells and 4T1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Wako,
Osaka, Japan) and Roswell Park Memorial Institute-1640 (Wako), respectively, supplemented
with 10% fetal bovine serum (Dainippon Sumitomo, Osaka, Japan) at 37˚C in a 5% CO2
incubator.

In vitro assays with B16-F1 and 4T1 cells
In vitro assays were performed using our methods, with some modifications [19]. Briefly, for
the inhibition study, B16-F1 and 4T1 cells were seeded into 24-well cell culture multiwell plates
at a density of 5×105 cells/well. Assays were conducted 24 hours after seeding. B16-F1 and 4T1
cells were pre-incubated with each type of medium including 1.0 υM α-MSH for 180 min at
37˚C. B16-F1 and 4T1 cells were incubated for 180 min with 111In-labeled 3-arm or 4-arm
DOTA-α-MSH.
For the accumulation study with 111In-labeled 3-arm and 4-arm DOTA-α-MSH, each well
was incubated with each type of medium for 10 min at 37˚C. Then, 20 kBq/well 111In-labeled
3-arm or 4-arm DOTA-α-MSH was added and incubated for 10, 30, 60, 120, and 180 min at
37˚C as the control condition. At the end of the incubation in the inhibition and accumulation
studies, each well was rapidly washed twice with 500 μL 0.5 M HEPES buffer. Cells were thensolubilized in 500 μL 0.1 M NaOH. The radioactivity that accumulated in B16-F1 and 4T1 cells

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20194 / 144-arm DOTA construct affects the biological distribution

was measured with a gamma counter (ARC-380; Hitachi-Aloka Medical, Tokyo, Japan).
B16-F1 and 4T1 cells were detached with trypsin, and the protein in the cells was measured
using a protein assay. All experimental conditions were examined with quadruplicate assays.

Stability analysis of 111In-labeled 3-arm and 4-arm DOTA-α-MSH in mice
All animal studies were conducted following approval by the Animal Care Committee ofKanazawa University (AP-122339). Fasted B16-F1-bearing C57BL6 male mice (5 weeks old)
were administered 111In-labeled 3-arm or 4-arm DOTA-α-MSH (3.7 MBq/mouse) via the tailvein. At 10, 30, 60, 120, and 180 min after injection, mice (n = 3 per time point) were euthanized with isoflurane, and blood and liver were collected and analyzed with thin layer chromatography (TLC). Briefly, 300 uL blood in a heparinized tube was centrifuged at 18,000 ×g at
4˚C for 5 min. A total of 30 uL perchloric acid was added to the supernatant, which was centrifuged again, after which the final supernatant was spotted onto the TLC plate as plasma.
Krebs-Ringer phosphate buffer (pH 7.4) was added to the liver samples, followed by homogenization with an ultrasonic homogenizer (SONIFIER250, Branson, MO, USA). Then, ethanol
was added to the homogenate to remove proteins, and the sample was centrifuged at 18,000 ×g
at 4˚C for 5 min. The final supernatant was spotted onto the reverse-phase TLC plate, and the
TLC spots were developed using 50 mM EDTA (pH 4–5) solvents. After development andcomplete drying, the TLC plates were cut into 21 fractions, and the radioactivity associatedwith each fraction was quantified using a γ-ray counter. The fraction ratios of 111In3+, 111Inlabeled 3-arm or 4-arm DOTA-α-MSH, and all metabolites were calculated by dividing theradioactive counts for each fraction by the total radioactivity counts. A stability study showedthat the fraction of 111In-labeled 3-arm or 4-arm DOTA-α-MSH remained the same at all
injection times.

Biological distribution and whole-body single photon emission computed
tomography (SPECT) imaging of 111In-labeled 3-arm and 4-arm DOTA-αMSH in B16-F1-bearing mice
C57BL6 male mice (5 weeks old) were transplanted with B16-F1 cells (5 × 105 cells/100 μL)
into the thigh of the mice [19]. The tumors appeared and gradually increased in size after
about 10 days. The mice were housed and continuously monitored each day for 2 weeks in a
12-hour light/12-hour dark cycle with free access to food and water. The tumor size reached8.7 ± 2.8 mm at 2 weeks after transplant of cells. Two mice with tumor sizes of 13 and 15 mm
were excluded from our experiments. Mice with a tumor size <10 mm were fasted with nofood overnight with water supplied ad libitum before experiments.
For biological distribution studies, B16-F1-bearing mice were administered 111In-labeled
3-arm or 4-arm DOTA-α-MSH via the tail vein (37 kBq/mouse). At 10, 30, 60, 120, and 180
min after injection, the mice were euthanized under isoflurane (n = 3 per time point), and thefollowing tissues were collected: brain, lung, heart, stomach, liver, small intestine, large intestine, kidney, blood, muscle, and B16-F1 melanoma. Tissues were weighed, and radioactivity
was quantified using an automated γ-ray counter to calculate the percent injected dose (%ID)
or percent injected dose per gram of tissue (%ID/g).
For SPECT imaging, 111In-labeled 3-arm or 4-arm DOTA-α-MSH was injected into the tailvein of four B16-F1-bearing mice in total (37 MBq/mouse). For SPECT imaging, acquisition
was started at 10 min after injection and continued every 10 min for 180 min. The data werereconstructed using the ordered subset expectation maximization method with 16 subsets andsix iterations including no scatter and attenuation correction. The voxel size was set to
0.8×0.8×0.8 mm. Post-reconstruction smoothing filtering was applied using a 1.0-mm

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20195 / 144-arm DOTA construct affects the biological distribution

Gaussian filter. Image displays were obtained using medical image data analysis software,
AMIDE (ver. 1.04). The coronal images were displayed as maximum intensity projections. In
these images, three to five regions of interest were placed over the kidney, muscle, and B16-F1
melanoma in which accumulation could be seen, and the time activity curve of each was
obtained as the %ID/g.

Statistical analysis
Data are presented as the mean and standard deviation (SD). P values were calculated using a
two-tailed Student’s t-test. Results were considered to be significant at P < 0.01.

Results
Specific activity, radiolabeling efficiency, and partition coefficients of
111
In-labeled 3-arm or 4-arm DOTA-α-MSH
Specific activities of both 111In-labeled 3-arm and 4-arm DOTA-α-MSH were about 1.2 MBq/
nM. With HPLC analysis, the retention times were 2.5–3.0 min for 111In3+, 14–17 min for
111
In-labeled 3-arm DOTA-α-MSH, and 20–22 min for 111In-labeled 4-arm DOTA-α-MSH.
Radiolabeling efficiencies of both 111In-labeled 3-arm and 4-arm DOTA-α-MSH were 90–95%. Non-carrier-added radiotracers for both compounds were obtained with a radio-chemical purity of >95% after purification. For TLC analysis of partition coefficients, the Rf values of
111
In-labeled 3-arm DOTA-α-MSH, 4-arm DOTA-α-MSH, and 111In3+ were 0.35–0.45, 0.40–
0.50, and 0.00, respectively. Partition coefficients were −0.28 ± 0.03 for 111In-labeled 3-arm
DOTA-α-MSH and −0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH.

In vitro assays with B16-F1 and 4T1 cells
Fig 2 shows the studies without and with the substrate of 1.0 μM α-MSH and 111In-labeled
3-arm and 4-arm DOTA-α-MSH in B16-F1 melanoma cells with high MC1-R expression and
4T1 breast cancer cells with low MC1-R expression measured with real-time qPCR. Accumulation of both 111In-labeled 3-arm and 4-arm DOTA-α-MSH was significantly inhibited by αMSH in B16-F1 cells, but the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower
than that of 111In-labeled 3-arm DOTA-α-MSH. In 4T1 cells, we found no significant accumulation of either radiotracer.
The accumulation of 111In-labeled 3-arm and 4-arm DOTA-α-MSH in B16-F1 cells isshown in Fig 3. 111In-labeled 4-arm DOTA-α-MSH was taken up early into the B16-F1 cells
and accumulated more than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation.

Stability analysis of 111In-labeled 3-arm and 4-arm DOTA-α-MSH in mice
Rf values of some metabolites were 0.10–0.30. Few metabolites were seen at 10 and 30 min
after injection (Table 1). At 60 min after injection, the fraction ratios of 111In-labeled 3-arm
DOTA-α-MSH were about 86.4%, 84.3%, and 85.7% in the plasma, liver, and kidney of themice, respectively, whereas the fraction ratios of 111In-labeled 4-arm DOTA-α-MSH wereabout 92.1%, 91.3%, and 93.3%, respectively. At 120 min after injection, the fraction ratios of111
In-labeled 3-arm DOTA-α-MSH were about 79.2%, 71.6%, and 78.5% in the plasma, liver,and kidney of the mice, respectively, whereas the fraction ratios of 111In-labeled 4-arm DOTAα-MSH were about 84.2%, 78.6%, and 84.5%, respectively. At 180 min after injection, the fraction ratios of 111In-labeled 3-arm DOTA-α-MSH were about 73.5%, 62.9%, and 74.3% in theplasma, liver, and kidney of the mice, respectively, whereas the fraction ratios of 111In-labeled
4-arm DOTA-α-MSH were about 80.8%, 76.2%, and 80.5%, respectively.

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Fig 2. In vitro assays of 111In-labeled 3-arm DOTA-α-MSH and 111In-labeled 4-arm DOTA-α-MSH in B16-F1 melanoma
cells and 4T1 breast cancer cells after 180 min of incubation in the presence or absence (control) of α-MSH. The
accumulation of both radiotracers in cells was significantly inhibited by α-MSH. In 4T1 cells with low MC1-R expression, no
significant accumulation and no inhibition effect were observed for either radiotracer. �� P < 0.01 vs. control. https://doi.org/
10.6084/m9.figshare.7697882.
https://doi.org/10.1371/journal.pone.0213397.g002

Fig 3. In vitro assays of 111In-labeled 3-arm DOTA-α-MSH (■) and 4-arm DOTA-α-MSH (●) in B16-F1
melanoma cells. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher
accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. �� P < 0.01 and � P < 0.05 between
111
In-labeled 3-arm DOTA-α-MSH and 4-arm DOTA-α-MSH. https://doi.org/10.6084/m9.figshare.7697885.
https://doi.org/10.1371/journal.pone.0213397.g003

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Table 1. Stability analysis of 111In-labeled 3-arm and 4-arm DOTA-α-MSH in plasma, liver, and kidney of mice by ex vivo.

111

In labeled 3-arm DOTA-α-MSH

111

In labeled 4-arm DOTA-α-MSH

Time (min)

10

30

60

120

180

Plasma (%)

96.1 ± 2.0

95.1 ± 1.8

86.4 ± 2.5

79.2 ± 2.2

73.5 ± 2.4

Liver (%)

95.7 ± 2.1

95.5 ± 1.9

84.3 ± 2.2

71.6 ± 4.5

62.9 ± 6.7

Kidney (%)

95.8 ± 2.0

95.9 ± 1.7

85.7 ± 2.8

78.5 ± 2.7

74.3 ± 4.1

Plasma (%)

96.0 ± 2.5

95.8 ± 2.6

92.1 ± 2.1

84.2 ± 2.2

80.8 ± 2.3

Liver (%)

95.1 ± 2.9

94.9 ± 2.1

91.3 ± 3.2

78.6 ± 4.9

76.2 ± 4.1

Kidney (%)

96.1 ± 2.0

95.8 ± 1.9

93.3 ± 3.5

84.5 ± 3.3

80.5 ± 2.8

All data are the mean ± standard deviation from triplicate measurements using three mice per time pointhttps://doi.org/10.6084/m9.figshare.7697815
https://doi.org/10.1371/journal.pone.0213397.t001

Biological distribution and whole-body SPECT imaging of 111In-labeled
3-arm and 4-arm DOTA-α-MSH in B16-F1-bearing mice
The biological distributions of 111In-labeled 3-arm DOTA-α-MSH (Table 2) and 4-arm
DOTA-α-MSH (Table 3) were obtained in B16-F1 melanoma-bearing mice. Radioactivity inthe liver, kidney, and blood rapidly increased immediately after injection and then gradually
decreased. In the lung and melanoma, the radioactivity reached a peak at 30–60 min after
injection. Very little radioactivity was present in other organs. In kidney, 111In-labeled 4-arm
DOTA-α-MSH provided significantly lower average accumulation (1.38-fold) than 111Inlabeled 3-arm DOTA-α-MSH at all acquisition time points, whereas 111In-labeled 4-arm
DOTA-α-MSH provided significantly higher average accumulation (1.32-fold) than 111Inlabeled 3-arm DOTA-α-MSH in B16-F1 melanoma. Whole-body SPECT images of B16F1-bearing mice were obtained for 111In-labeled 3-arm (a) and 4-arm DOTA-α-MSH (b) at
170–180 min after injection (Fig 4). The main accumulation was found in kidney and B16-F1
tumors in images. In particular, accumulation of 111In-labeled 4-arm DOTA-α-MSH was
higher than that of 111In-labeled 3-arm DOTA-α-MSH in B16-F1 melanoma. In other organs,little accumulation of 111In-labeled 3-arm DOTA-α-MSH and 111In-labeled 4-arm DOTA-αMSH was observed.
Table 2. Biological distribution of 111In-labeled 3-arm DOTA-α-MSH.
Organ(%ID/g or %ID/organ)

10 min30 min60 min

120 min180 min

Brain

0.04

±

0.01

0.05

±

0.02

0.05

±

0.01

0.03

±

0.00

0.02

±

Lung

0.51

±

0.15

0.66

±

0.24

0.31

±

0.09

0.27

±

0.09

0.10

±

0.04

Heart

0.74

±

0.15

0.66

±

0.13

0.46

±

0.15

0.14

±

0.06

0.06

±

0.02

Stomach�

0.06

±

0.03

0.10

±

0.03

0.12

±

0.03

0.11

±

0.05

0.12

±

0.03

Liver

2.30

±

1.04

1.15

±

0.92

1.12

±

0.86

1.23

±

0.47

0.72

±

0.15

Small intestine�

0.04

±

0.03

0.08

±

0.02

0.09

±

0.03

0.12

±

0.03

0.10

±

0.03

Large intestine

0.03

±

0.01

0.08

±

0.02

0.09

±

0.03

0.05

±

0.03

0.04

±

0.03

Kidney

23.55

±

3.63

18.42

±

5.57

11.87

±

5.40

8.42

±

4.27

7.07

±

3.02

Blood

3.11

±

0.84

1.91

±

0.45

0.89

±

0.22

0.21

±

0.06

0.05

±

0.02

Muscle

0.21

±

0.07

0.20

±

0.08

0.15

±

0.08

0.09

±

0.04

0.05

±

0.02

Melanoma

1.71

±

0.64

1.81

±

0.45

2.49

±

0.62

3.36

±

0.90

3.51

±

0.78

�

0.00

%ID/g indicates percent injected dose per gram of tissue.
�%ID/organ was calculated from %ID per organ. Values are the average ± standard deviation.

Values are the mean ± standard deviation obtained from three mice per time point.
https://doi.org/10.6084/m9.figshare.7698227
https://doi.org/10.1371/journal.pone.0213397.t002

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20198 / 144-arm DOTA construct affects the biological distribution

Table 3. Biological distribution of 111In-labeled 4-arm DOTA-α-MSH.
10 minOrgan(%ID/g or %ID/organ)

30 min60 min

120 min180 min

Brain

0.04

±

0.01

0.03

±

0.01

0.03

±

0.01

0.02

±

0.00

0.01

±

0.00

Lung

0.42

±

0.12

0.52

±

0.14

0.22

±

0.08

0.13

±

0.04

0.06

±

0.01

Heart

0.55

±

0.11

0.46

±

0.10

0.38

±

0.08

0.14

±

0.03

0.05

±

0.01

Stomach�

0.09

±

0.02

0.09

±

0.03

0.08

±

0.02

0.05

±

0.01

0.05

±

0.01

Liver

2.52

±

0.73

1.45

±

0.74

1.18

±

0.69

0.90

±

0.33

0.61

±

0.12

Small intestine�

0.05

±

0.01

0.04

±

0.01

0.03

±

0.01

0.02

±

0.00

0.02

±

0.00

Large intestine�

0.03

±

0.01

0.03

±

0.02

0.04

±

0.01

0.03

±

0.01

0.03

±

0.01

Kidney

17.14

±

4.12

13.33

±

3.87

8.12

±

3.11

6.31

±

2.11

5.19

±

1.82

Blood

4.03

±

0.93

2.02

±

0.54

1.01

±

0.28

0.11

±

0.04

0.03

±

0.00

Muscle

0.10

±

0.03

0.12

±

0.05

0.08

±

0.02

0.04

±

0.00

0.03

±

0.00

Melanoma

2.22

±

0.65

2.25

±

0.63

3.34

±

0.93

4.52

±

1.01

4.83

±

1.03

%ID/g indicates percent injected dose per gram of tissue.
�%ID/organ was calculated from %ID per organ. Values are the average ± standard deviation.

Values are the mean ± standard deviation obtained from three mice per time point.
https://doi.org/10.6084/m9.figshare.7698230
https://doi.org/10.1371/journal.pone.0213397.t003

Table 4 summarizes melanoma-to-kidney and melanoma-to-muscle ratios. The averagemelanoma-to-kidney ratios for 111In-labeled 3-arm DOTA-α-MSH were 0.07, 0.10, 0.21, 0.40,
and 0.50 at 10, 30, 60, 120, and 180 min after injection, respectively. For 111In-labeled 4-arm
DOTA-α-MSH, the ratios were 0.13, 0.17, 0.41, 0.72, and 0.93, respectively. The average melanoma-to-blood ratios for 111In-labeled 3-arm DOTA-α-MSH were 0.55, 0.95, 2.80, 16.00, and
65.00 at 10, 30, 60, 120, and 180 min after injection. For 111In-labeled 4-arm DOTA-α-MSH,
the ratios were 0.55, 1.11, 3.31, 41.09, and 155.81, respectively. The average melanoma-to-muscle ratios for 111In-labeled 3-arm DOTA-α-MSH were 8.14, 9.05, 16.60, 37.33, and 66.23 at 10,30, 60, 120, and 180 min after injection. For 111In-labeled 4-arm DOTA-α-MSH, the ratios
were 22.20, 18.75, 41.75, 113.00, and 155.81, respectively.

Discussion
In this study, the 4-arm DOTA construct was newly applied for conjugation to α-MSH to evaluate specific activity, radiolabeling efficiency, MC1-R affinity, stability, and tumor accumulation in melanoma imaging. Although specific activity and radiolabeling efficiency of 111Inlabeled 4-arm DOTA-α-MSH were not much different from those of 3-arm DOTA-α-MSH,
which is the generally used DOTA construct, specific activity was quite low compared to labeling with other MC1-R analogues [14,20]. Because the partition coefficients of 111In-labeled
3-arm and 4-arm DOTA-α-MSH were negative, these radiotracers are water soluble and areusually renally excreted. We observed high renal accumulation and excretion of 111In-labeled
3-arm and 4-arm DOTA-α-MSH (Fig 4). However, the lipophilicity of 111In-labeled 4-arm
DOTA-α-MSH was slightly closer to zero than that of 111In-labeled 3-arm DOTA-α-MSH
according to the partition coefficients. We estimated that the lipophilicity resulted in a slightly
different distribution between 111In-labeled 3-arm DOTA-α-MSH and 111In-labeled 4-arm
DOTA-α-MSH (Tables 2 and 3).
In the inhibition study (Fig 2), because accumulation of both 111In-labeled 3-arm and4-arm DOTA-α-MSH was significantly inhibited by the substrate, α-MSH, in B16-F1 cellswith high MC1-R expression, and not inhibited in 4T1 cells with low MC1-R expression, both

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 20199 / 144-arm DOTA construct affects the biological distribution

Fig 4. Whole-body SPECT images of B16-F1-bearing mice under 2.0% isoflurane anesthesia injected with 37 MBq 111In-labeled 3-arm DOTA-α-MSH (a) or
111
In-labeled 4-arm DOTA-α-MSH (b) at 170–180 min after injection. The highest accumulation was seen in the tumors and kidney for both radiotracers. We
observed that the tumor accumulation of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. https://doi.org/10.
6084/m9.figshare.7697897.
https://doi.org/10.1371/journal.pone.0213397.g004

radiotracers bound to MC1-R. Although the inhibition rate of 111In-labeled 4-arm DOTA-αMSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH at 180 min after incubation,
the amount of inhibition of 111In-labeled 4-arm DOTA-α-MSH (mean 3.28) was higher thanthat of 111In-labeled 3-arm DOTA-α-MSH (mean 2.67) according to the calculation from Figs

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 201910 / 14

4-arm DOTA construct affects the biological distribution

Table 4. Melanoma-to-organ ratios of 111In-labeled 3-arm and 4-arm DOTA-α-MSH in B16-F1-bearing mice.
Time (min)Melanoma-to-kidney

Melanoma-to-blood

Melanoma-to-muscle

3-arm DOTA-α-MSH

4-arm DOTA-α-MSH

3-arm DOTA-α-MSH

4-arm DOTA-α-MSH

3-arm DOTA-α-MSH

4-arm DOTA-α-MSH

100.07 ± 0.03

0.13 ± 0.07�

0.55 ± 0.13

0.55 ± 0.14

8.14 ± 1.44

22.20 ± 3.35��

30

0.10 ± 0.04

0.17 ± 0.11��

0.95 ± 0.30

1.11 ± 0.28�

9.05 ± 1.38

18.75 ± 4.45��

0.21 ± 0.04

��

2.80 ± 0.58

��

16.60 ± 3.28

41.75 ± 6.38��

��

37.33 ± 4.23

113.00 ± 7.33��

��

66.23 ± 4.21

155.81 ± 8.63��

60
120
180

0.40 ± 0.09
0.50 ± 0.10

0.41 ± 0.15

��

0.72 ± 0.18

��

0.93 ± 0.15

16.00 ± 3.21
65.00 ± 4.64

3.31 ± 0.35

41.09 ± 4.78

155.81 ± 12.93

All data were mean ± standard deviation measured in four mice.
��P < 0.01 and

P < 0.05 between 111In labeled 3-arm DOTA-α-MSH and 4-arm DOTA-α-MSH.
https://doi.org/10.6084/m9.figshare.7698239

�https://doi.org/10.1371/journal.pone.0213397.t004

2 and 3. Therefore, nonspecific accumulation of 111In-labeled 4-arm DOTA-α-MSH (mean0.92) was higher than that of 111In-labeled 3-arm DOTA-α-MSH (mean 0.23). This moderate
nonspecific accumulation is estimated to be caused by lipophilicity of 111In-labeled 4-arm
DOTA-α-MSH. In Fig 3, the time-activity curve of 111In-labeled 4-arm DOTA-α-MSH wassignificantly higher than that of 111In-labeled 3-arm DOTA-α-MSH in B16-F1 melanoma
cells. In the in vitro study, the high accumulation of 111In-labeled 4-arm DOTA-α-MSH may
be caused by the effect of lipophilicity and high affinity for MC1-R in B16-F1 melanoma cells.
Moderate stability of 111In-labeled 3-arm and 4-arm DOTA-α-MSH was found at all time
points (Table 1). The stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of111
In-labeled 3-arm DOTA-α-MSH. In addition, these stabilities were relatively higher thananother peptide radiotracer conjugated to 3-arm DOTA [21]. One reason may be that eightcoordinate complexes of 111In-labeled 4-arm DOTA conjugation are generally more stable
than seven-coordinate complexes of 111In-labeled 3-arm DOTA conjugation [22, 23].
In the biological distribution of 111In-labeled 3-arm (Table 2) and 4-arm DOTA-α-MSH(Table 3) and whole-body SPECT imaging (Fig 4), accumulation of 111In-labeled 4-arm
DOTA-α-MSH was significantly higher than that of 111In-labeled 3-arm DOTA-α-MSH in
B16-F1 melanoma. This also supports the similar results from the in vitro study in Fig 3. 111Inlabeled 4-arm DOTA-α-MSH showed significantly lower renal accumulation than 111Inlabeled 3-arm DOTA-α-MSH at all acquisition times. The number of carboxy groups affects
electric charges in the whole body and changes the whole-body distribution of peptide radiotracers [24]. Thus, the effect of electric charges may yield lower renal accumulation. For peptide radiotracers, reducing renal accumulation is important. Behr et al. showed that systemic
administration of cationic amino acids reduces renal reabsorption and accumulation of peptide radiotracers [25]. However, amino acid infusion may change the whole-body distribution
and increase background accumulation in muscle, blood, and kidney etc. On the other hand,
111
In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma accumulation than111
In-labeled 3-arm DOTA-α-MSH. This may be because of the slightly higher lipophilicity,
significantly higher MC1-R affinity, and relatively higher stability of 4-arm DOTA-α-MSH
compared to 3-arm DOTA-α-MSH. If renal accumulation is reduced and tumor accumulation
is increased using other peptides conjugated to the 4-arm DOTA construct compared with the
3-arm DOTA construct, the 4-arm construct will be a simple and useful DOTA construct forlabeling of peptide radiotracers.
The melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios (Table 4)of 111In-labeled 4-arm DOTA-α-MSH were significantly higher than those ratios of 111In-

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 201911 / 14

4-arm DOTA construct affects the biological distribution

labeled 3-arm DOTA-α-MSH. However, these ratios were relatively lower than those of other111
In-labeled DOTA peptides [12] because we selected the native α-MSH peptide hormone toevaluate the chemical properties between the 3-arm DOTA and 4-arm DOTA constructs. We
selected native α-MSH as a good peptide to evaluate improvement of chemical properties
using the 4-arm DOTA construct because 3-arm DOTA-α-MSH has relatively low yield,
MC1-R affinity, and stability compared to other 3-arm DOTA-conjugated peptides [14]. The
IC50 values of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were 4.8 ± 0.5 nM and 5.3 ± 0.4
nM, respectively. These are similar to or better than those of numerous 111In-labeled α-MSH
analogues (0.9–78.6 nM) [12]. Therefore, utilization of α-MSH for comparison of 111In-labeled
3-arm and 4-arm DOTA peptides was considered appropriate. However, when other peptides
are applied for 111In labeling of the 4-arm DOTA construct, melanoma-to-tissue ratios will
improve more than other 111In-labeled DOTA peptides with the 3-arm DOTA construct.
Thus, 4-arm DOTA peptides will be effective for tumor diagnosis because of their slightly
higher lipophilicity, higher MC1-R affinity, and relatively higher stability compared to 3-arm
DOTA peptides. https://doi.org/10.6084/m9.figshare.7698239
For internal radiation therapy, we can use 4-arm DOTA constructs with 225Ac labelinginstead of 111In. 225Ac-labeled 4-arm DOTA peptides are estimated to form eight-coordinate
complexes similar to 111In-labeled 4-arm DOTA peptides because the chemical characteristics
and properties of 225Ac are similar to those of 111In [26]. Therefore, 225Ac-labeled 4-arm
DOTA peptides may provide higher melanoma accumulation, better internal radiation therapy, and relatively lower renal accumulation, similar to 111In-labeled 4-arm DOTA peptides.

Conclusion
111

In-labeled 4-arm DOTA-α-MSH yields higher tumor accumulation and lower renal accumulation than 111In-labeled 3-arm DOTA-α-MSH because the chemical properties of the
4-arm DOTA construct include slightly higher lipophilicity, significantly higher MC1-R affinity, and relatively higher stability. Therefore, 4-arm DOTA constructs provide better chemical
properties for peptide radiotracers than 3-arm DOTA constructs.

Acknowledgments
The authors would like to thank Mikie Ohtaki and the other laboratory staff members of Kanazawa University, National Institutes of Health, and The University of Texas MD Anderson
Cancer Center.

Author Contributions
Conceptualization: Masato Kobayashi, Kohshin Washiyama, Leo G. Flores, 2nd, Ryuichi
Nishii, Keiichi Kawai.
Data curation: Masato Kobayashi.
Formal analysis: Masato Kobayashi.
Funding acquisition: Masato Kobayashi.
Investigation: Masato Kobayashi, Toshitaka Kato, Masaaki Ihara, Asuka Mizutani, Kodai
Nishi, Leo G. Flores, 2nd, Keiichi Kawai.
Methodology: Masato Kobayashi, Toshitaka Kato, Kohshin Washiyama, Masaaki Ihara,
Asuka Mizutani, Kodai Nishi, Leo G. Flores, 2nd.
Project administration: Masato Kobayashi, Asuka Mizutani, Kodai Nishi, Ryuichi Nishii.

PLOS ONE | https://doi.org/10.1371/journal.pone.0213397 March 22, 201912 / 14

4-arm DOTA construct affects the biological distribution

Software: Masato Kobayashi.
Supervision: Masato Kobayashi, Kohshin Washiyama, Leo G. Flores, 2nd, Ryuichi Nishii,
Keiichi Kawai.
Validation: Masato Kobayashi.
Visualization: Masato Kobayashi, Masaaki Ihara.
Writing – original draft: Masato Kobayashi.
Writing – review & editing: Keiichi Kawai.

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